Genome Replication in Independent of Cdc6 and an Origin of Replication.

The initiation of DNA replication is typically tightly regulated by proteins that form initiation complexes at specific sequences known as replication origins. In Archaea and Eukaryotes, Cdc6, a near-universally conserved protein binds and facilitates the origin-dependent assembly of the replicative apparatus. TK1901 encodes Cdc6 in but, as we report here, TK1901 and the presumed origin of replication can be deleted from the genome of this hyperthermophilic Archaeon without any detectable effects on growth, genetic competence or the ability to support autonomous plasmid replication.

Structure of histone-based chromatin in Archaea.

Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome.

The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis.

Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In , a 5'-to-3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for INS-ssociated uclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex.

Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis.

Background: Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.

Results: Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements.

Archaeal nucleosome positioning in vivo and in vitro is directed by primary sequence motifs.

Background: Histone wrapping of DNA into nucleosomes almost certainly evolved in the Archaea, and predates Eukaryotes. In Eukaryotes, nucleosome positioning plays a central role in regulating gene expression and is directed by primary sequence motifs that together form a nucleosome positioning code. The experiments reported were undertaken to determine if archaeal histone assembly conforms to the nucleosome positioning code.

Thermococcus kodakarensis has two functional PCNA homologs but only one is required for viability.

Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively.

Archaeal DNA polymerase D but not DNA polymerase B is required for genome replication in Thermococcus kodakarensis.

Three evolutionarily distinct families of replicative DNA polymerases, designated polymerase B (Pol B), Pol C, and Pol D, have been identified. Members of the Pol B family are present in all three domains of life, whereas Pol C exists only in Bacteria and Pol D exists only in Archaea. Pol B enzymes replicate eukaryotic chromosomal DNA, and as members of the Pol B family are present in all Archaea, it has been assumed that Pol B enzymes also replicate archaeal genomes.

An archaeal histone is required for transformation of Thermococcus kodakarensis.

Archaeal histones wrap DNA into complexes, designated archaeal nucleosomes, that resemble the tetrasome core of a eukaryotic nucleosome. Therefore, all DNA interactions in vivo in Thermococcus kodakarensis, the most genetically versatile model species for archaeal research, must occur in the context of a histone-bound genome. Here we report the construction and properties of T.

Thermococcus kodakarensis encodes three MCM homologs but only one is essential.

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukaryotes. In eukaryotes, this complex is an assembly of six different but related polypeptides (MCM2-7) but, in most archaea, one MCM protein assembles to form a homohexameric complex. Atypically, the Thermococcus kodakarensis genome encodes three archaeal MCM homologs, here designated MCM1-3, although MCM1 and MCM2 are unusual in having long and unique N-terminal extensions.

Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production.

Hydrogen (H₂) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H₂ and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H₂ production for reductant disposal.

A novel DNA nuclease is stimulated by association with the GINS complex.

Chromosomal DNA replication requires the spatial and temporal coordination of the activities of several complexes that constitute the replisome. A previously uncharacterized protein, encoded by TK1252 in the archaeon Thermococcus kodakaraensis, was shown to stably interact with the archaeal GINS complex in vivo, a central component of the archaeal replisome. Here, we document that this protein (TK1252p) is a processive, single-strand DNA-specific exonuclease that degrades DNA in the 5' → 3' direction.

Preliminary crystallography confirms that the archaeal DNA-binding and tryptophan-sensing regulator TrpY is a dimer.

TrpY regulates the transcription of the metabolically expensive tryptophan-biosynthetic operon in the thermophilic archaeon Methanothermobacter thermautotrophicus. TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87, c = 147 Å, and diffracted to 2.

Affinity purification of an archaeal DNA replication protein network.

Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His(6)-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His(6)-tagged proteins, stable complexes assembled in vivo have been isolated directly from clarified cell lysates and the T. kodakarensis proteins present have been identified by mass spectrometry.

Deletion of switch 3 results in an archaeal RNA polymerase that is defective in transcript elongation.

Switch 3 is a polypeptide loop conserved in all multisubunit DNA-dependent RNA polymerases (RNAPs) that extends into the main cleft of the RNAP and contacts each base in a nascent transcript as that base is released from the internal DNA-RNA hybrid. Plasmids have been constructed and transformed into Thermococcus kodakaraensis, which direct the constitutive synthesis of the archaeal RNAP subunit RpoB with an N-terminal His(6) tag and the Switch 3 loop either intact (wild-type) or deleted (DeltaS3). RNAPs containing these plasmid-encoded RpoB subunits were purified, and, in vitro, the absence of Switch 3 had no negative effects on transcription initiation or elongation complex stability but reduced the rate of transcript elongation.

Thermococcus kodakarensis genetics: TK1827-encoded beta-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology.

Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second beta-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95 degrees C and from pH 8 to 9.5, and have a functional half-life of approximately 7 min at 100 degrees C.

Archaeal intrinsic transcription termination in vivo.

Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter and a beta-glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5'-TTTTTTTT (T(8)) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (t(mcrA)) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression approximately 90%, but this required only the oligo(T) trail sequence and not the inverted-repeat and loop region.

Archaeal aIF2B interacts with eukaryotic translation initiation factors eIF2alpha and eIF2Balpha: Implications for aIF2B function and eIF2B regulation.

Translation initiation is down-regulated in eukaryotes by phosphorylation of the alpha-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2alpha interacts with a subcomplex of eIF2B formed by the three regulatory subunits alpha/GCN3, beta/GCD7, and delta/GCD2, blocking the GDP-GTP exchange activity of the catalytic epsilon-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO(2) fixation.

The Fur iron regulator-like protein is cryptic in the hyperthermophilic archaeon Thermococcus kodakaraensis.

Archaea, which regroup organisms with extreme living conditions, possess many predicted iron-containing proteins that may be metabolically critical; however, their need for iron remains poorly documented. In this report, iron acquisition mechanisms were investigated in the hyperthermophilic archaeon Thermococcus kodakaraensis. Thermococcus kodakaraensis requires iron for its growth and possesses many putative iron uptake systems, including several ATP-binding cassette-like transporters and two FeoAB-like receptors, showing that this organism shares similar features with bacteria.

Nanoarchaeal origin of histone H3?

NEQ288, one of two archaeal histones in Nanoarchaeum equitans, has a unique four-residue insertion that closely resembles an insertion in the eukaryotic histone H3 lineage. NEQ288 bound DNA but did not compact DNA in vitro in the absence of NEQ348, the second N. equitans archaeal histone.

Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature-sensitive.

All archaeal genomes encode RNA polymerase (RNAP) subunits E and F that share a common ancestry with the eukaryotic RNAP subunits A43 and A14 (Pol I), Rpb7 and Rpb4 (Pol II), and C25 and C17 (Pol III). By gene replacement, we have isolated archaeal mutants of Thermococcus kodakarensis with the subunit F-encoding gene (rpoF) deleted, but we were unable to isolate mutants lacking the subunit E-encoding gene (rpoE). Wild-type T.

Archaeal chromatin proteins histone HMtB and Alba have lost DNA-binding ability in laboratory strains of Methanothermobacter thermautotrophicus.

Alignments of the sequences of the all members of the archaeal histone and Alba1 families of chromatin proteins identified isoleucine residues, I19 in HMtB and I39 in MtAlba, in Methanothermobacter thermautotrophicus, at locations predicted to be directly involved in DNA binding. In all other HMfB family members, residue 19 is an arginine (R19), and either arginine or lysine is present in almost all other Alba1 family members at the structural site equivalent to I39 in MtAlba. Electrophoretic mobility shift assays revealed that recombinant HMtB and MtAlba do not bind DNA, but variants constructed with R19 and R39, respectively, bound DNA; and whereas MtAlba(I19) did not bind RNA, MtAlba(R19) bound both single stranded RNA and tRNA.

Shuttle vector expression in Thermococcus kodakaraensis: contributions of cis elements to protein synthesis in a hyperthermophilic archaeon.

Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli have been constructed. Plasmid pTN1 from Thermococcus nautilis was ligated to the commercial vector pCR2.1-TOPO, and selectable markers were added so that T.

TrpY regulation of trpB2 transcription in Methanothermobacter thermautotrophicus.

TrpY binds specifically to TRP box sequences upstream of trpB2, but the repression of trpB2 transcription requires additional TrpY assembly that is stimulated by but not dependent on the presence of tryptophan. Inhibitory complex formation is prevented by insertions within the regulatory region and by a G149R substitution in TrpY, even though TrpY(G149R) retains both TRP box DNA- and tryptophan-binding abilities.

Polarity in archaeal operon transcription in Thermococcus kodakaraensis.

An in vivo archaeal gene reporter system has been established based on TK1761, a gene that encodes a nonessential beta-glycosidase in Thermococcus kodakaraensis. Following the introduction of nonsense codons into promoter-proximal genes, polarity in operon expression in this archaeon has been established by both microarray hybridization assays and a reporter gene expression system.